Helicases comprise a large group of enzymes, from classical to pseudo-helicases, which play important roles in genome maintenance. The pseudo-helicases have been the subject intense investigation in biological processes ranging from cancer, chromatin remodeling, to long-range communication between distant DNA sites. While some of pseudo-helicases are bone-fide motors or translocases that consume hundreds of ATP molecules to processively move on the DNA/RNA, others are turning out to be molecular switches that hydrolyze just a few ATPs to switch structural states for long-range diffusion. These molecular switches are important in processes ranging from nucleotide excision repair to mismatch repair, but their mechanism of action remains mysterious. The Type III restriction enzymes (REs) offer the ideal system to investigat pseudo-helicase activity because all of the enzymatic functions are integrated in the same holoenzyme complex and no additional protein cofactors are required. We propose here a set of experiments combining X-ray crystallography with state-of-the-art single-molecule and ensemble measurements to elucidate how, EcoP15I, a prototype of the Type III RE family, transitions from one state to another for long-lived sliding on DNA. In Aim 1, we wil derive the first 3-D structural information on EcoP15I. In addition to the native Ecop15I/DNA complex, we will determine structures in the presence of ADP and ATP analogues, as well as structure of the enzyme in synaptic or collision complex. The proposed structural studies are the first for a Type III restriction enzyme, and only the second for a helicase bound to double-stranded DNA. In aim 2, we will derive a kinetic framework for the interpretation of structural results. Guided by the structure, we will perform single molecule and ensemble fluorescence resonance energy transfer measurements of EcoP15I dynamics during interaction with DNA and ATP. We will take advantage of a specially built magnetic tweezers-total internal reflection fluorescence (MT-TIRF) microscope that can visualize single fluorescently- labeled proteins sliding along DNA stretched within a magnetic field. This will be complemented by millisecond time resolution fluorescent assays using stopped flow, as well as new Biacore-based DNA dissociation assays. Together, the proposed real-time assays will complement the structural studies and provide unprecedented new details on the reaction pathway of an ATP-dependent molecular switch in DNA metabolism.